This revised proposal extends a current K08 award to study a novel target of myocardial preconditioning, and builds on 5 years of studies in chick cardiomyocytes that demonstrate (a) significant oxidant injury at reperfusion following ischemia, (b) hypoxic preconditioning (PC) which attenuates this injury, and (c) a burst of reactive oxygen species (ROS) at reperfusion which originate from an NAD(P)H oxidase. We propose to advance these studies in a recently established murine cardiomyocyte system to further establish a mechanism by which PC attenuates reperfusion oxidants generated by this oxidase. These murine cardiomyocytes also exhibit hypoxic preconditioning against reperfusion ROS and allow for study of transgenic mouse cardiomyocytes. The central hypothesis of this proposal is that classical PC, by opening mitochondrial KATP channels, increases NO generation at reperfusion and thereby inhibits NAD(P)H oxidase activity, superoxide generation, and subsequent reperfusion injury. We propose two specific aims, testing whether: (1) PC or mitochondrial KATP channel openers given at reperfiision increase transient calcium release from the mitochondria, activate constitutive nitric oxide synthase (cNOS), and thereby attenuates reperfusion ROS and its associated injury, and whether this protection can be abrogated by addition of -NADPH and -NADH and (2) Alterations of NAD(P)H oxidase, using murine cardiomyocytes from NAD(P)H knockout mice, will attenuate reperfusion ROS and confer preconditioning-like protection, which will not be effected by inhibitors of mitochondrial KATP channels or NOS. This work will clarify how PC blocks an important source of ROS, and could improve postresuscitation care following cardiac arrest and other ischemia/reperfusion diseases such as myocardial infarction and stroke.